Multivalent felv-infected feline vaccine

ABSTRACT

A multivalent FeLV-infected feline vaccine composed of (1) a small but immunologically effective amount of inactivated feline leukemia virus; (2) a small but effective amount of an inactivated virus selected from the group consisting of Feline Rhinotracheitis Virus, Feline Calici Virus and Feline Panleukopenia Virus; and (3) a pharmaceutically acceptable immunologica adjuvant. Components (1) and (2) may be inactivated by any of the known techniques. The inactivated feline leukemia virus must be from subgroup A and is the Rickard isolate of the virus designated FeLV-A R1 . This combination vaccine is effective in preventing Viremia, leukemia-associated syndromes, and deaths in cats caused by Feline Leukemia Virus, Feline Rhinotracheitis Virus, Feline Calici Virus and Feline Panleukemia Virus infections.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of co-pending, commonly assigned application Ser. No. 915,088 filed Oct. 3, 1986, now abandoned.

BACKGROUND OF THE INVENTION

Feline leukemia virus (FeLV) is a retrovirus consisting of three subgroups designated A, B, and C. The FeLV subgroups were identified initially by viral interference and cross-neutralization. Sarma & Log, Virology (1973) 54:160-169; see also, Russell & Jarrett, Int'l J. Cancer (1978) 21: 768-778; and Jarrett (1980) in Feline Leukemia Virus-Developments in Cancer Research Vol. 4, Hardy et al., eds., Elsevier North Holland, Inc., pp. 473-479. Feline leukemia virus is discussed also in Jarrett & Russell, Int'l J. Cancer (1978) 21: 466-472; Sarma et al., J. Nat'l Cancer Inst. (1978) 60: 871-874; and Schaller & Olsen Infect. Immun. (1975) 12: 1405-1510.

FeLV is infectious in cats. It is responsible for a number of diseases, including lymphoplastic or aplastic anemia, myelosuppression, thymic atrophy, and thrombocytopenia, as well as reproductive failure, e.g., abortion fetal resorption, and stillbirths. Neoplastic manifestations of FeLV infection, such as lymphosarcoma, account for only a small portion of the morbidity and mortality caused by FeLV. FeLV infection in cats, however, also causes suppression of the immune system, thereby exposing the animal to opportunistic infections by a variety of microorganisms.

Vaccines have been developed for FeLV. See, e.g., U.S. Pat. No. 4,332,793 to Olson, U.S. Pat. No. 4,264,587 to Pedersen et al., U.S. Pat. No. 4,117,112 to Jarrett et al., U.S. Pat. No., 4,086,134 to Jarrett et al., U.S. Pat. No. 4,034,081 to Jarrett et al., U.S. Pat. No. 3,966,907 to Jarrett et al., and Belgian Patent Application R 64 417 DG to the University of Glasgow.

In particular, a FeLV vaccine may be prepared from inactivated FeLV virus and from envelope glycoprotein (gp70), the antigen with which it is thought protective antibody must react. Such viral material generally may be produced by culturing infected feline cell lines, but it has been impossible to predict whether a particular feline cell line will produce vital material of appropriate quality in quantities sufficient to be useful for commercial production of FeLV vaccines. Moreover, a cell line which satisfactorily produces viral material for a given strain of FeLV may not produce adequate protection against other strains or may produce vital material that for other reasons is ineffective in vaccine formulations.

Thus, there is a continuing need to develop new systems for producing FeLV viral material that can be used in formulating commercially useful FeLV vaccines. It should also be noted that FeLV viral material is useful in the preparation of diagnostic reagents, and thus, new cell lines for its production are needed for that purpose as well.

There is also a continuing need for development of multivalent vaccines for FeLV which also successfully vaccinate for other viruses which commonly infect cats. Such multivalent vaccines must not only be effective in development of the desired immune response but the admixture of the various inactivated viruses must be such that each is compatible with the other and the adjuvant.

The objectives of the present invention include development of safe and effective monovalent and multivalent FeLV vaccines useful for vaccination of cats against FeLV virus and certain other common, infectious viruses of cats.

SUMMARY OF THE INVENTION

Our earlier invention provided for Crandell Feline Kidney (CRFK) cells infected with a Rickard isolate of FeLV, i.e., FeLV-A_(R1), with the infected cells being capable of producing FeLV virus in amounts sufficient for formulating safe and effective FeLV vaccines. This invention improves on our earlier one by providing a multivalent or combination FeLV vaccine.

This invention also provides for a process for producing FeLV vital material, which process comprises infecting CRFK cells with an FeLV virus, such as FeLV-A_(R1), cultivating the infected CRFK cells, recovering the vital material, and using it to provide multivalent FeLV vaccines.

Surprisingly, it has now been found that the FeLV-infected Crandell cell line of the subject invention stably yields high titers of FeLV which can be used in safe and efficacious multivalent FeLV vaccines. Particularly, it produces 10 to 100 times higher titers of gp70 than do normal feline embryo cells.

DETAILED DESCRIPTION OF THE INVENTION

The feline cell line employed in the present invention is the Crandell Feline Kidney (CRFK) line obtained from the American Type Culture Collection (ATCC CCL94). CRFK cells are described in Crandell et al., In Vitro (1973) 9:176-185.

The CRFK cell line is infected with a Rickard isolate of FeLV, i.e., FeLV-A_(R1). Upon culturing, the infected cell line, designated ACFL, becomes chronically infected and produces viral material at a rate of at least 10³ to 10⁷ focus-forming units per milliliter. The FeLV virus produced by the cells exhibits a titer of 10⁵ preferably 10⁶ focus-forming units per milliliter (FFU/ml) in one to three weeks, and at least 2×10⁶ preferably 3×10⁶ FFU/ml thereafter.

On the other hand, when infected with another Rickard isolate, FeLV-A_(R2), the CRFK cells produced less viral material, and the vital material produced was less effective in vaccine formulations. Similarly, viral material produced from another FeLV-A_(R1) -infected cell line, AK-D cells, which satisfactorily produce viral material when infected with the Sarma strain of FeLV, was inadequate for formulating vaccines.

The viral material produced by the ACFL cell line may be used in a variety of vaccine formulations within the scope of this invention. The viral material is virulent for cats and must be inactivated before it is used to prepare vaccines.

The viral material may be inactivated by conventional means which maintain immunogenicity. Means of inactivation are known in the art. Particular means of inactivation which have found use herein include heat, formalin, binary ethyleneimine (BEI), ozone, and psoralen. See U.S. Pat. Nos. 4,332,793; 4,264,587; 4,117,112; 4,034,081; and 3,996,907. Preferably either BEI or psoralen inactivation will be employed. In the psoralen method the live virus is combined in an appropriate medium with the psoralen. The amount of psoralen should be sufficient to provide for a substantially complete inactivation of the virus upon irradiation with long wave ultraviolet radiation. See U.S. Pat. No. 4,545,987.

In accord with the present improvement invention the same FeLV inactivation procedure and techniques are employed as employed in our earlier invention of the monovalent vaccine. Thus, the techniques as described in our earlier filed application are wholly applicable to the present improved vaccine as well. This present vaccine, that is the improved one, involves a multivalent or combination vaccine which vaccinates for FeLV and at least one other common feline viral infection.

The other viruses which may be inactivated by use of the same general procedures described herein and included in the multivalent improved vaccine include one or more of the following viruses: Feline Rhinotracheitis Virus (FVR); Feline Calici Virus (FCV); and Feline Panleukopenia Virus (FPV). Each of these three additional viruses are common viral infections of cats. FVR provides disease symptoms similar to the common human cold. FCV provides disease symptoms of swollen and/or watering eyes and common coughing. FPV virus is a systematic infection of cats and may often be fatal.

Surprisingly, any combination of these inactivated viruses are compatible with the basic monovalent FeLV vaccine of the parent application. They are compatible not only from the standpoint of being compatible with the adjuvant but neither interferes with development of the desired antigenic response of the other. Thus, the vaccine of the present invention includes not only the basic inactivated FeLV vaccine but also one or more or all of the additional inactivated viruses, i.e. Feline Rhinotracheitis Virus, Feline Calici Virus, and Feline Panleukopenia Virus.

The dosage levels in the combination or multivalent vaccines of the present invention should generally be within the following ranges. For FeLV the preferred range is from about 6.5×10⁶ FFU equivalents per dose to 1×10⁷ FFU equivalents per dose. For FVR the preferred dosage level is from about 10⁶.5 TCID₅₀ equivalents or more per dose to about 10⁷ TCID₅₀ equivalents per dose. For FCV the preferred dose level ranges from about 10⁵.6 TCID₅₀ equivalents or more per dose to about 10⁶ equivalents per dose. For FPV the preferred range is from about 10⁵.1 TCID₅₀ equivalents or more per dose to 10⁶ equivalents per dose.

The vaccines may include a physiologically-acceptable carrier, for example, an inert carrier such as deionized water, phosphate-buffered saline, saline, or the like. The carrier may also include a physiologically-acceptable adjuvant, such as a mineral oil, vegetable oil, mineral salt, or an immunopotentiator, such a muramyl dipeptide. Such carriers and adjuvants are well known in the art.

Typically, the concentration of the FeLV virus in the vaccine formulation, either separated from or within the infected cell, will be from about 10² to about 10⁹ FFU/ml. If needed, the virus may be conveniently concentrated using commercially available equipment. The total dosage generally is at least about 10² FFU per dose, more commonly at least about 10⁵ per dose. It generally will not exceed about 10⁹ FFU per dose.

Usually, a specific dosage at a specific site will be administered in a volume in the range from about 0.1 ml to 4 ml, where the total volume administered will range from 0.5 ml to 8 ml. The number of injections and their temporal spacing may be varied, but one to three injections at one to three week intervals are usually effective.

The vaccine may be administered subcutaneously, intramuscularly, or intraperitoneally. Conveniently, the prepared vaccines will be packaged in small vials holding sufficient vaccine for one dose and having a septum for inserting a hypodermic needle.

The subject invention is further described by reference to the following example. These examples are not intended to limit the scope of the invention; rather, they are presented merely to facilitate the practice of the invention by those of ordinary skill in the art.

EXAMPLE 1

The production of FeLV virus and gp70 was compared in (1) FeLV-A_(R1) infected CRFK cells, (2) FeLV-A_(R2) infected CRFK cells, and (3) FeLV-A_(R1) infected AK-D cells as follows:

Infection and Culturing

CRFK cells (ATCC CCL94) were seeded at a density of approximately 5×10⁶ cells into a 75 cm² tissue culture flask in 12 ml of MEN₉₅ F^(i) ₅ growth medium and incubated overnight at 37° C. (MEN₉₅ F^(i) ₅ is Eagle's Minimum Essential Medium with Earle's salts and nonessential amino acids supplemented with 5% heat-inactivated fetal bovine serum). At 20 hours postseeding the growth medium was removed from the cell monolayer, and 2 ml of overlay medium (MEN containing 25 ug/ml of DEAE-Dextran) was added. The cells then were incubated for 1 hour at 37° C. After incubation, the overlay medium was removed, and the cell monolayer was rinsed two times with 10 ml of MEN₉₅ F^(i) ₅.

The cell monolayer then was overlayed with 2 ml of FeLV-A_(R1) having titer of approximately 2.5×10⁴ FFU/ml. The virus was allowed to absorb for 2 hours at 37° C. The multiplicity of infection (MOI) was 0.01. Following the 2 hour adsorption period, cells were fed with 12 ml MEN₉₅ F^(i) ₅ growth medium and incubated at 37° C. The initial flask of CRFK cells infected with FeLV-A_(R1) was designated O-ACFL.

Following the initial infection, the medium was changed at three-day intervals. Confluency generally was reached on the seventh day following initial infection, i.e., day seven post-infection (p.i.), at which time the cells were subcultured at a split ratio of 1:10 in a new flask. This same process was carried out at seven-day intervals for 5 weeks, the subsequent flasks being designated 1-ACFL, 2-ACFL, etc.

The same procedure described above for FeLV-A_(R1) infected CRFK cells also was used to infect CRFK cells with FeLV-A_(R2) and to infect AK-D cells with FeLV-AR₁.

At 7-day intervals p.i., samples of medium containing FeLV-A were withdrawn. The samples were titrated and assayed for p27 as described below with the results shown in Table 1 below, thereby confirming that the cell lines were chronically infected.

Recovery of Vital Material

At confluency, 250 ml MEN₉₅ F^(i) ₅ were added to each of 10 roller bottles of fifth passage ACFL cells (5-ACFL). After 72 hours the supernatant was harvested, pooled, and frozen with 10% DMSO and stored at -85° C. Fresh medium was added to the roller bottles and at 144 hours the supernatant was again harvested, pooled, and frozen. The harvests were thawed, pooled, and concentrated 10-fold to a final volume of 500 ml using a Millipore Corporation Pellicon ultrafiltration cassette (100,000 MW exclusion).

The same procedure was used to concentrate virus harvested from fifth passage, FeLV-A_(R2) infected CRFK cells (5-CRFK). The vital material produced by the FeLV-A_(R1) infected AK-D cells was not recovered because the production levels of viral material were too low to be of interest for formulating vaccines.

The pooled and concentrated viral materials from the 5-ACFL and 5-CRFK cell lines were titrated and assayed for p27 as described below. They were also assayed for gp70 and tested for reverse transcriptase activity, again as described below.

Virus Titration: Determination of Focus Forming Units

Standard test cells (clone 81) were plated into 6-cm plastic petri dishes at 2×10⁵ cells/dish in growth medium (McCoy's 5A+15% heat-inactivated fetal calf serum) and incubated in a humidified CO₂ incubator. Approximately 20 hours later the medium was withdrawn and 1 ml of DEAE-Dextran (DEAE-D) (25 ug/ml) in growth medium was added to each dish. The cells were then incubated at 37° C. After 30 minutes the DEAE-D medium was withdrawn, and each dish was washed once with 2 ml of growth medium.

Virus inocula of 0.2 to 1.0 ml per dish were applied, the dishes were incubated, with occasional shaking, for 30 minutes at 37° C., and then 5 ml of growth medium was added to each dish. On day 3 p.i., 3 ml of growth medium was added. On day 7 p.i., all growth medium was removed and replaced with 5 ml of fresh growth medium. On day 10 p.i., 3 ml of fresh growth medium was added.

Foci were read on day 12 p.i. The foci typically are 0.5 to 2 mm in diameter and consist of piled, small, dark, round cells on a dense monolayer. Generally, no retraction of the monolayer occurs in the focus area.

Further details of this procedure are set forth in Fischinger et al., J. Virol (1974) 14:177-179.

p27 Assay

Supernatants from infected cultures were assayed for FeLV group specific (p27) antigen by an ELISA (Leukassay*F, commercially available from Pitman Moore) according to the manufacturer's specified procedure.

gp70 Assay

Supernatants from infected cultures were assayed for FeLV gp70 by an indirect ELISA. A microtiter plate coated with polyclonal goat anti-FeLV gp70 antibody (NCI Repository) was used as the capturing solid phase. The antibody used to detect virus bound to the plate was a mouse anti-FeLV gp70 monoclonal antibody (AGRI). The second antibody was a peroxidase-conjugated goat anti-mouse immunoglobulin (Boehringer Mannhelm Biochemicals). The substrate used was tetramethylbenzidine in citrate buffer.

Quantitation of gp70 was based on comparison with a standard sample of purified virus. The standard virus sample was prepared by banding virions in a preformed linear 15-60% sucrose gradient by velocity sedimentation in an ultracentrifuge using standard methods. Test and standard samples were diluted serially and assayed by ELISA. The optical density of each dilution point was determined. The titer of the test sample relative to the virus standard was computed at 50% of the maximum optical density of the test sample.

Reverse Transcriptase Assay

Cell culture supernatants were tested for reverse transcriptase activity by the following procedure: Fifty microliters of 2X cocktail were added to each 50 ul aliquot of cell culture supernatant. (2X Cocktail contains 80 mM Tris-HCl1 (pH 8.2), 120 mM KCl, 1.2 mM MnAcetate, 1 OD₂₆₀ poly-A, 0.01 OD₂₆₀ oligodT₁₂₋₁₈, 0.1 mM TTP, 0.4% NP-40, 3.0 mM dithiothreitol, 0.1 uCi/ul ³ H-TTP in deionized water). The mixture was incubated for 30 minutes at 37° C., whereupon the reaction was stopped, and the DNA was precipitated by adding 0.2 ml of 0.1M sodium polyphosphate and 0.3 ml of 20% cold trichloroacetic acid (TCA) to each tube. The tubes then were incubated for 30 minutes at 0° C. Samples were collected on 2.4 cm Whatman GF/A filters. Sample tubes were washed twice with cold 5% TCA. Filters were washed twice with cold 5% TCA followed by cold 100% ethanol and then dried. The radioactivity of the viral-specific DNA was counted in 7 ml scintillation fluid in a Beckman Instruments model LS-1800 scintillation counter.

Results

The titration and various assay results are reported in Table 1. As shown therein, the ACFL line produced virus of higher titer, sooner after infection, and continued to produce virus and vital antigen at a higher sustained level than did CRFK cells infected with FeLV-A_(R2) or AK-D cells infected with FeLV-A_(R1). The infected AK-D cells did not produce virus of sufficiently high titer to be useful in vaccine production. This result was unexpected, because AK-D cells have been used successfully to produce quantities of the Sarma strain of FeLV-A sufficient for preparing experimental vaccines.

                                      TABLE 1                                      __________________________________________________________________________     Titration and Assay Data From Cell Lines Infected With FeLV-A.sub.R                        Week                                                                               Titer    p27                                                                               ELISA                                                                              gp70  RT activity                              System                                                                               Passage                                                                              P.1 FFU/ml                                                                               Dil.                                                                              Plate                                                                             O.D.                                                                               ELISA cmp/50 pl                                __________________________________________________________________________     ACFL  0-ACFL                                                                               1   ND    Neat                                                                              +   1.385                                                                             ND    ND                                             1-ACFL                                                                               2   ND    Neat                                                                              +  2.37                                                                               ND    ND                                             2-ACFL                                                                               3   1.15 × 10.sup.6                                                                Neat                                                                              +  2.96                                                                               ND    ND                                             3-ACFL                                                                               4   2.25 × 10.sup.6                                                                Neat                                                                              +  3.10                                                                               ND    ND                                             4-ACFL                                                                               5   3.75 × 10.sup.6                                                                Neat                                                                              +  2.87                                                                               ND    ND                                             5-ACFL                                                                               6                                                                        5L Pool   1.73 × 10.sup.6                                                                Neat                                                                              +  3.26                                                                                1:95**                                                                              ND                                             10X Conc. 2.18 × 10.sup.7                                                                Neat                                                                              +  3.20                                                                               1:16  108.590                                                        1:10                                                                              +  2.10                                                                               ND    ND                                       CRFK- 0-CRFK                                                                               1   ND    Neat                                                                              +   1.997                                                                             ND    ND                                       FeLV-AR.sub.2                                                                        1-CRFK                                                                               2   ND    Neat                                                                              +  2.92                                                                               ND    ND                                             2-CRFK                                                                               3   3.20 × 10.sup.5                                                                Neat                                                                              +  3.01                                                                               ND    ND                                             3-CRFK                                                                               4   3.75 × 10.sup.5                                                                Neat                                                                              +  3.08                                                                               ND    ND                                             4-CRFK                                                                               5   5.25 × 10.sup.5                                                                Neat                                                                              +  2.69                                                                               ND    ND                                             5-CRFK                                                                               6                                                                        5L Pool   4.90 × 10.sup.5                                                                Neat                                                                              +  3.25                                                                                 1:112**                                                                            ND                                             10X Conc. 4.89 × 10.sup.6                                                                Neat                                                                              +  3.29                                                                               1:20   61.200                                                        1:10                                                                              +   1.814                                                                             ND    ND                                       AKD-  0-AK-D                                                                               1   ND    Neat                                                                              +   0.421                                                                             ND    ND                                       FeLV-AR.sub.1                                                                        1-AK-D                                                                               2   ND    Neat                                                                              +   0.996                                                                             ND    ND                                             2-AK-D                                                                               3   3.20 × 10.sup.4                                                                Neat                                                                              +   1.597                                                                             ND    ND                                             3-AK-D                                                                               4   1.85 × 10.sup.4                                                                Neat                                                                              +   0.994                                                                             ND    ND                                             4-AK-D                                                                               5   7.75 × 10.sup.3                                                                Neat                                                                              +   0.980                                                                             ND    ND                                       __________________________________________________________________________      *Positive control  3.04; Negative control  0.09                                **Equivalent dilution of virus standard as determined by graphic               interpolation.                                                                 ND  Not Done                                                             

EXAMPLE 2

The production of FeLV-A_(R1) viral material in eighth-passage, chronically infected ACFL cells and in eleventh-passage, chronically infected normal feline embryo cells was compared. Infection, culturing, titration, and assaying were performed as described above in Example 1. The results are reported below in Table 2. As shown therein, the ACFL line produced more virus and gp70 viral antigen than did the normal feline embryo cells infected with the same virus isolate.

                  TABLE 2                                                          ______________________________________                                         Titration and Assay Data From ACFL and Normal Feline                           Embryo Cells Infected with FeLV-A.sub.R1                                                                          gp70                                               Titer            p27  ELISA ELISA RT activity                           System FFH/ml    Dil.   Plate                                                                               O.D.* ARB*  cmp/50 pl                             ______________________________________                                         ACFL    1.0 × 10.sup.6                                                                    Neat   +    3.26  21    55,828                                                 1:10   +    3.22                                                               1:100  +    2.46                                              Normal 2.14 × 10.sup.5                                                                    Neat   +     1.250                                                                               2.7   11,204                                Feline                                                                         Embryo                                                                         ______________________________________                                          *ACFL: Positive control = 3.17; Negative control  0.088                        Feline Embryo: Positive control = 3.01; Negative control = 0.087               **ARB = One ARB unit is an arbitrary measurement equivalent to 1/100 of a      standard preparation of purified FeLVA (Rickard).                        

EXAMPLE 3

The effectiveness of vaccine formulations derived from the ACFL and FeLV-A_(R2) infected CRFK cell lines was compared as follows:

Vaccine Formulations

A first vaccine formulation was prepared from the pooled, concentrated vital material produced by the ACFL cells of Example 1. The concentrated virus preparation was inactivated by 0.2% formalin for approximately 48 hours at room temperature. The vaccine contained approximately 10⁷ FFU of virus suspension and an oil-in-water adjuvant in a 1:1 ratio.

A second vaccine formulation was prepared in the same manner from the pooled, concentrated virus produced by the FeLV-A_(R2) infected CRFK cells of Example 1. It contained approximately 2.4×10⁶ FFU of virus.

Feline Subjects

Eighteen cats (minimal disease cats, Liberty Labs, Liberty Corner, N.J.), nine males and nine females, 14 to 15 weeks of age, were divided into three groups of six cats each. Groups I and II were vaccinated intramuscularly twice with a 3-week interval with 1 ml, respectively, of the first and second vaccine formulations. Group III cats served as unvaccinated controls. All cats were challenged oronasally two weeks after the second inoculation with 10⁶ FFU of live FeLV-A_(R1).

Blood samples were taken weekly. The samples were assayed for FeLV antigens, by p27 ELISA as described above, and by indirect immunofluorescence, as described below. They also were tested by the virus isolation protocol described below.

Immunofluorescence Assay (IFA)

Smears of blood from vaccinated cats were tested for the presence of FeLV antigens in blood cells by indirect immunofluorescence- The first antibody was goat anti-FeLV p27 (NCI Repository); the second antibody was fluorescein-conjugated rabbit anti-goat IgG (Copper Biomedical/Cappel Laboratories). Slides were examined on a Zeiss Photomicroscope III equipped for epifluorescence.

Virus Isolation Protocol

The cats were kept in isolation. Blood samples were aseptically collected. Serum was separated by centrifugation and filtered through 0.2 um filter discs (e.g., Gelman Acrodiscs). Lymphocytes were separated by density centrifugation by diluting 1 to 1.5 ml EDTA-treated blood with 1 to 1.5 ml phosphate buffered saline (PBS), layering the suspension onto 2.5 ml Ficoll-Paque (Pharmacia), and centrifuging the tubes at 800×g for 30 minutes. Cells banded at the interface were harvested and washed once in PBS. Lymphocytes were resuspended in 0.2 ml of autologous serum.

An indicator cell culture was prepared by growing AK-D feline lung cells (ATCC CCL150) in F12K medium supplemented with 15% heat-inactivated fetal bovine serum and antibiotics (100 units/ml Penicillin and 100 ug/ml Streptomycin).

The AK-D cells were harvested by trypsin treatment. Approximately 5×10⁵ cells were seeded into each 25 cm² flask with 4 ml medium. The cultures were incubated at 37° C. with 5% CO₂ for one day. The medium was removed, and the resuspended lymphocytes (0.2 ml) were inoculated onto the AK-D cell monolayer. Control cultures were inoculated with PBS. After the inoculum was adsorbed for 1 hour, 5 ml of medium was added. The medium was changed the next day. Thereafter, the cells were cultured at 37° C. with 5% CO₂, fed on day 3 or 4, and subcultured on day 7. The cultures were maintained for up to six weeks.

Cell culture supernatant was collected for FeLV p27 antigen assay before feeding (3 to 5 days post-inoculation) and also 3 to 5 days post-subculturing. The collected supernatant then was tested for the presence of FeLV p27 by ELISA (Leukassay*F, Pitman Moore) according to the manufacturer's specified procedure. Cultures that gave two consecutive positive assays were considered "virus isolation positive"; cultures that remained negative for six weeks were considered "virus isolation negative".

The validity of this protocol was confirmed by testing supernatants for the presence of infectious retrovirus using the clone-81 virus assay described above in Example 1.

Results

At eleven weeks post-challenging none of the Group I cats were viremic by IFA or p27 ELISA assays. In contrast 4 of 6 Group II cats and 5 of 6 Group III cats were viremic by both assays. At post-challenge week five, 5 or 6 cats in Group II and 5 of 6 cats in Group III were virus isolation positive whereas 0 of 6 cats in Group I were virus isolation positive.

Virus was not isolated from the blood of the two cats that were positive by ELISA the day before challenge. Since virus isolation is generally a more sensitive test than ELISA, the ELISA results are considered to be spurious. It also is not known whether the failure of the vaccine produced from FeLV-A_(R2) grown in CRFK cells may be attributed to the effective dose, the characteristics of the antigen(s), and/or other factors.

In summary, the chronically infected ACFL line produced more virus and gp70 viral antigen than did the other cell line-virus isolate combinations studied. Virus from ACFL produced an efficacious vaccine, while virus from CRFK cells infected with a different isolate did not yield an efficacious vaccine. The ACFL line constitutes an improved system for producing high titer FeLV virus and the virus produced is useful in formulating vaccines. The ACFL virus also would be useful for production of diagnostic reagents.

These results are summarized below in Table 3.

                                      TABLE 3                                      __________________________________________________________________________     Summary of Serum FeLV ELISA and Virus Isolatation Data                         From Cats Vaccinated with Inactivated FeLV Vaccines                            Groups I and II Vaccinated: Day 0 and Day 21                                   Groups I, II & III Challenged: Day 35                                          Group                                                                              Test D-15                                                                              D-21                                                                              D-34                                                                              D-49                                                                              D-55                                                                              D-64                                                                              D-70                                                                              D-77                                                                              D-84                                                                              D-91                                                                              D-98                                                                               D-105                                                                              D-112                           __________________________________________________________________________     I   Serum                                                                               0/6                                                                               0/6                                                                               1/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               1/6                                                                               0/6                                                                               0/6                                                                               0/6 0/6 0/6                                 ELISA                                                                          1FA     0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6 0/6 0/6                                 Virus      0/1         0/6          0/6*                                       Isol.                                                                      II  Serum                                                                               0/6                                                                               0/6                                                                               1/6                                                                               0/6                                                                               4/6                                                                               4/6                                                                               5/6                                                                               5/6                                                                               5/6                                                                               4/6                                                                               4/6 4/6 4/6                                 ELISA                                                                          1FA     0/6                                                                               0/6                                                                               0/6                                                                               1/5                                                                               4/6                                                                               4/6                                                                               5/6                                                                               5/6                                                                               4/6                                                                               4/6 5/6 4/6                                 Virus      0/2         5/6                                                     Isol.                                                                      III Serum                                                                               0/6                                                                               0/6                                                                               0/6                                                                               0/6                                                                               3/6                                                                               5/6                                                                               5/6                                                                               5/6                                                                               5/6                                                                               5/6                                                                               5/6 5/6 5/6                                 1FA     0/6                                                                               0/6                                                                               0/6                                                                               3/6                                                                               4/6                                                                               5/6                                                                               5/6                                                                               5/6                                                                               5/6                                                                               5/6 6/6 6/6                                 Virus                  6/6                                                     Isol.                                                                          Baseline                                                                   __________________________________________________________________________      Data reported as number of positive per group                                  *assay in progress                                                             D = day                                                                  

All publications cited in this application are indicative of the level of skill of those skilled in the art to which this invention pertains. Each publication is individually incorporated herein by reference in the same location and to the same extent as if each publication had been individually incorporated by reference.

EXAMPLE 4-MULTIVALENT VACCINES

A four-way multivalent vaccine was prepared that included killed Felines leukemia Virus, killed Feline Rhinotracheitis Virus, killed Feline Calici Virus and killed Feline Panleukopenia Virus. In preparing the vaccine, that is an assembly of the serial, one or more lots each having inactivated virus, with each inactivated as described below were transferred to suitable sterile vessels. Adjuvant, as described below was added and the mixture was vigorously agitated for 60 minutes until the product was ready for fill. The components of the assembled product were as follows:

    ______________________________________                                                            Volume   Total                                              Component          Per Dose Volume                                             ______________________________________                                         Feline Leukemia Virus                                                                             0.05 ml  17,000 ml                                          Feline Rhinotracheitis Virus                                                                      0.17 ml   5,780 ml                                          Feline Calici Virus                                                                               0.17 ml   5,780 ml                                          Feline Panleukopenia Virus                                                                        0.16 ml   5,440 ml                                          Adjuvant           0.50 ml  17,000 ml                                          Total for Fill     1.50 ml  51,000 ml                                          ______________________________________                                    

For this vaccine, the Feline Leukemia Virus employed was the Feline Leukemia Virus prepared as described in example 1 and inactivated as shown in example 3.

The FVR, FCV, and FPV viruses were grown in Eagles's Minimum Essential Medium (MEM) with the addition of up to 5% bovine serum. Lactalbumin hydrolysate (LAH) at 0.5% and gentamicin at 30 micrograms per milliliter were also added to the growth medium. The FVR, FCV, and FPV cells at a concentration of approximately 1×10⁵ cells per milliliter were suspended in the growth medium. The cultures incubated from two to seven days at approximately 36° C. in roller bottles.

The FVR, FCV, and FPV viral fluids were harvested after the incubation period and immediately inactivated.

The virus inactivation of the FVR, FCV, and FPV was by use of binary ethyleneimine (BEI). Particular, BEI was added to the pooled viruses to achieve a final concentration of 0.005 molar BEI. The volume of BEI added was calculated by the following formula:

    ml 0.2 M BEI=0.02632×(ml pooled virus)

Inactivation was allowed to proceed at from 25° C. to 36° C. with mixing for a period of from one to three days.

After inactivation, the BEI is neutralized with Sodium Thiosulfate (Na₂ S₂ O₃) Solution 50%. A sufficient volume of 50% Na₂ S₂ O₃ is added to the virus to achieve a final molarity of 0.05M. The volume of Na₂ S₂ O₃ added to neutralize the BEI is calculated by the formula:

    ml 50% Na.sub.2 S.sub.2 O.sub.3 =0.01634×(ml inactivated virus)

In preparing the multivalent vaccine composition of the total vaccine included a preservative and an adjuvant. The preservative was a 10% merthiolate solution added to the inactivated virus to achieve a final concentration of 0.01%. The adjuvant was an oil and water emulsion containing a copolymer.

In testing for safety and efficacy of the multivalent vaccine, the vaccine as prepared above was administered to many cats in a 1.5 ml dose to each cat by intramuscular inoculation. It was repeated in three to five weeks. The cats were thereafter challenged with live FeLV, FVR, FCV, and FPV. Results of the challenge show that the cats were successfully immunized and the cats remained healthy. The vaccine, that is the multivalent vaccine, was thus shown to be an aid in preventing Viremia, leukemia-associated syndromes, and deaths caused by Feline Leukemia Virus, Feline Rhinotracheitis Virus, Feline Calici Virus, and Feline Panleukopenia Virus infections.

There was no evidence of incompatibility of each of the inactivated viruses in either stability of the total vaccine or from the standpoint of each interfering with development of the desire to antigenic response of the other. It can therefore be seen that the multivalent feline leukemia vaccine was successful for developing the desired immune response in cats. 

What is claimed is:
 1. A multivalent Feline Leukemia Virus vaccine comprising:an immunogenic response producing amount of inactivated Rickard isolate feline Leukemia Virus designated FeLV-A_(R1) ; an effective amount of at least one inactivated virus selected from the group consisting of Feline Rhinotracheitis Virus, Feline Calici Virus, and Feline Panleukopenia Virus; and a pharmaceutically acceptable immunologic adjuvant.
 2. The multivalent vaccine of claim 1 in which the multivalent vaccine contains each of the following viruses: Feline Rhinotracheitis Virus, Feline Calici Virus, and Feline Panleukopenia Virus.
 3. The multivalent vaccine of claim 1 wherein the amount of Feline Leukemia Virus is within the range of 6.5×10⁶ TCID₅₀ to 1×10⁷ TCID₅₀.
 4. The multivalent vaccine of claim 1 wherein the amount of Feline Rhinotracheitis Virus is from about 10⁶.5 TCID₅₀ to about 10⁷ TCID₅₀.
 5. The multivalent vaccine of claim 1 wherein the amount of Feline Calici Virus is from about 10⁵.6 TCID₅₀ to about 10⁶ TCID₅₀.
 6. The multivalent vaccine of claim 1 wherein the amount of Feline Panleukopenia Virus is from about 10⁵.1 TCID₅₀ to about 10⁶ TCID₅₀.
 7. The multivalent virus vaccine of claim 1 wherein the FeLV-A_(R1) is formalin inactivated.
 8. The multivalent vaccine of claim 1 wherein the Feline Rhinotracheitis Virus, Feline Calici Virus and Feline Panleukopenia Virus have been inactivated with binary ethyleneimine.
 9. A method of immunizing cats comprising administering to a cat a multivalent vaccine containinga) an immunologically effective amount of inactivated Rickard isolate Feline Leukemia Virus designated FeLV-A_(R1) in combination with b) an immunologically effective amount of at least one inactivated viral material selected from the group consisting of Feline Rhinotracheitis Virus, Feline Calici Virus, and Feline Panleukopenia Virus and c) a nontoxic pharmaceutically acceptable immunologic adjuvant.
 10. The method of claim 9 wherein said multivalent vaccine is administered intramuscularly.
 11. The method of claim 9 wherein said vaccine is administered subcutaneously.
 12. The method of claim 9 wherein said vaccine is parenterally administered. 